ABSTRACT Recently a new bacterial second messenger termed cyclic di-adenosine monophosphate(c-di-AMP) has been identified and shown to play vital roles in diverse bacterial cellular processes. C-di-AMP is essential in many Gram-positive pathogens including Staphylococcus aureus and Streptococcus pneumoniae. We and others have found that c-di-AMP is not essential in Streptococcus mutans, an important etiological agent of dental caries (cavity), but regulates cariogenic biofilm formation, bacterial stress responses, and dynamic polymicrobial interactions that are crucial for S. mutans fitness and virulence. Little is known about the role of c-di-AMP signaling pathways that modulate distinct and conserved virulent properties found in S. mutans and other pathogens. In this proposal, we use S. mutans as a model to elucidate virulence regulation mediated by this emerging signaling molecule. Inactivation of the gene coding for c-di-AMP producing enzyme reduced bacterial colonization and virulence in a rat model of dental caries, demonstrating a critical role of c-di-AMP in S. mutans virulence. Moreover, c-di-AMP-mediated virulence networks are integrated into a key response regulator VicR- modulated signaling through a newly identified c-di-AMP binding receptor (CabPA). These novel findings led us to hypothesize that c-di-AMP regulates biofilm formation and other virulence properties via multiple new pathways, which represent major potential therapeutic targets to develop novel and selective anti-virulence compounds. Two specific aims are proposed to test the hypothesis: 1) To elucidate c-di-AMP and its receptor CabPA mediated signaling pathways that modulate the biofilm formation and other virulence properties; 2) To develop new small molecule compounds that modulate S. mutans virulence by targeting the c-di-AMP producing enzyme. The proposal would allow us to identify new signaling components and unknown dynamic interactions in c-di-AMP-mediated pathways responsible for the biofilm formation, oxidative stress, and bacterial competitiveness, and uncover potential therapeutic targets, which would facilitate the future development of new small molecule compounds that are amenable for drug discovery. The proposal tests a new hypothesis that links c-di-AMP signaling to VicR-mediated expression of a variety of virulence genes through a distinct c-di-AMP receptor, and documents non-essential nature of c-di-AMP signaling in S. mutans, and explores the translational potential using anti-virulence strategy. Successful completion of this application will have a direct impact on public health as dental caries and other infectious conditions are widespread due to antibiotic resistance and lack of effective treatment options. Development of anti-S. mutans virulence strategy in the proposal is also relevant to devise species-specific anti-virulence strategy targeting significant pathogens such as S. aureus and S. pneumoniae, which should open a new venue to the design of new anti-infectious agents to combat microbial infection and antibiotic resistance.